The invention relates to a mammalian cell cycle protein, p55CDC, DNA sequences encoding same, antibodies specific for the protein, a method for producing the protein and methods for modulating cell division by controlling the levels or activity of p55CDC or p55CDC-associated protein complexes.
The eukaryotic cell cycle has a growth phase and a reproductive phase, the latter composed of the chromosome cycle and the centrosome cycle which intersect in the establishment of the mitotic apparatus (for review, see 47). The profound morphologic changes which result in mitosis are accompanied by a cascade of phosphorylation and dephosphorylation events. In mammalian cells, different complexes of kinases and their associated regulatory proteins control progression through discrete steps of the cell cycle (for review, see 60, 67). While all eukaryotic cells use similar mechanisms to regulate progression through the stages of the cell cycle, it is clear that unique combinations of regulatory cyclins, kinases and phosphatases are responsible for cell- and organism-specific patterns of cell division (18, 51, 52).
A variety of kinases have been identified which control the crucial transitions through the cell cycle. The most well characterized is the p34cdc2 protein, which has been identified in all eukaryotic cells which have been examined (3, 16, 20, 28, 40, 41, 42, 59, 76). In addition, many other kinases which have homology to p34cdc2 and, like p34cdc2 fluctuate in activity during the cell cycle, have been described (48, 60). Other types of kinases have also been shown to vary in activity at different stages of the cell cycle, and have been proposed to play a role in control of cell division, although they share little or no homology with p34cdc2. These include the MAP kinases, and the MEK kinases which regulate MAP kinase activity (for review, see 11). In addition, a novel kinase has been identified in the fungus Aspergillus nidulans, the NIMA kinase, which is required to initiate mitosis (53-55). A mammalian kinase, Nek1, which has homology to the NIMA kinase, has been found in mouse, where it is expressed at high levels in gonadal tissues and may be required for meiosis (43).
As mentioned above, the activity of many of these kinases is regulated by their association with one or more cyclins. The cyclins are homologous with one another within a conserved region termed the cyclin box (44). The fluctuations in activity of the cyclin dependent kinases during the cell cycle result from differential association with newly synthesized cyclins, which are then degraded at specific transition points in the cell cycle. However, not all cyclins demonstrate the same degree of fluctuation during the cell cycle; for example, levels of the D type cyclins do not oscillate as dramatically during the cell cycle as the A and B type cyclins. In addition, a recently described cyclin, the mcs2 cyclin of S. pombe, shows no variation in level during the cell cycle, nor does the novel kinase activity associated with the mcs2 cyclin oscillate (49).
Experiments in yeast have defined a number of other cell division cycle (Cdc) proteins which are also crucial for the orderly progression of the cell cycle, although the functions of many of these proteins have not been precisely defined (34). Two of these proteins, the products of the CDC20 and CDC4 genes, have been proposed to be elements of the mitotic spindle or segregational apparatus (32). The cdc20 temperature sensitive mutants arrest in mitosis at the non-permissive temperature, after the formation of a complete short spindle and nuclear migration to the neck between the mother cell and a large bud (6). It has been proposed that the Cdc20 protein is directly required for chromosomal movement (56). In addition, the Cdc20 protein is required for modulation of microtubule structure, either by promoting microtubule disassembly (1, 65) or by altering the surface of the microtubules, and is also required for microtubule-dependent processes other than mitosis (65).
The CDC4 gene of S. cerevisiae (33), is essential for the initiation of DNA synthesis. Cells carrying a conditional-lethal, temperature-sensitive mutation in cdc4 arrest division at the non-permissive temperature, and the cells have a termination phenotype of multiple buds, a single nucleus, and duplicated spindle pole bodies connected by a bridge structure (6). CDC4 also appears to be required for karyogamy and sporulation (21, 68, 71). While the mechanism of action of the Cdc4 protein is still unknown, subcellular localization studies in yeast have demonstrated that it is associated with the nucleoskeleton (7). The appearance of the duplicated spindle pole bodies has been proposed to indicate that the CDC4 gene product is required for separation of the bodies and formation of the completed spindle (6, 75). It has recently been demonstrated that removal of the centrosome (the equivalent of the spindle pole body in higher eukaryotes) from mammalian cells uncouples the growth cycle from the reproductive cycle, indicating that cell division requires the presence of centrosomes to establish the bipolar mitotic spindle (45).
It is an object of the present invention to identify one or more proteins involved in regulation of the cell cycle, wherein said proteins may be targets for compounds which modulate the cell cycle. A novel protein, termed p55CDC has been identified. mRNA encoding p55CDC was ubiquitously present in all cell lines examined, as well as in embryonic tissue, placenta and adult hematopoietic tissues, but was not detected in cells induced to differentiate and cease cell division. The deduced amino acid sequence of human p55CDC demonstrates regions of homology with the S. cerevisiae Cdc20 and Cdc4 proteins within the Gxcex2-repeats found in the carboxy terminal half of these three proteins. Expression of p55CDC appears to be crucial for cell division in mammalian cells. p55CDC is phosphorylated in cycling cells. Immune complexes precipitated by a polyclonal antiserum to p55CDC have a kinase activity which fluctuates during the cell cycle, although p55CDC itself does not appear to be an endogenous substrate of the kinase activity.
The invention relates to a novel mammalian protein, p55CDC, which is essential for cell division. It has been found that p55CDC is expressed in actively proliferating cells while expression is not detected in slowly dividing or quiescent cells. Transfection of antisense p55CDC cDNA into CHO cells resulted in the isolation of only those cells having a compensatory increase in p55CDC mRNA having the sense orientation.
DNA sequences encoding biologically active p55CDC are also provided by the invention. DNA sequences include rat (SEQ. ID NO: 1) and human (SEQ. ID NO: 3) p55CDC and DNA hybridizing to rat or human p55CDC, or to a fragment thereof, wherein the hybridizing DNA encodes biologically active p55CDC. Also provided for are vectors containing p55CDC DNA sequences and host cells transformed or transfected with said vectors. A method of producing a p55CDC polypeptide comprising culturing transformed or transfected host cells such that p55CDC is expressed is also included
p55CDC polypeptides of the invention will preferably form a complex with one or more host proteins such that the complex has cell-cycle dependent kinase activity. The kinase activity of p55CDC complexes will fluctuate during the cell cycle.
A method for modulating cell division is also encompassed by the invention, wherein the method comprises introducing into a cell (e.g. a tumor cell) a compound which modulates the kinase activity of p55CDC complexes. Modulation of p55CDC associated kinase activity may involve an increase or decrease in activity at certain periods during the cell cycle which in turn may lead to alterations in timing or specificity of p55CDC-associated kinase activity. In a preferred embodiment, cell division is inhibited by exposure to compounds which interfere with p55CDC complex formation.